One of the most striking cell cycle related biochemical changes yet observed in mammalian cells is the extensive phosphorylation of histones H1 and H3 at mitosis. This phosphorylation results from the action of a specific protein kinase, and it correlates closely with chromosome condensation, the breakdown of the nuclear envelope, and the shut-off of transcription. However, its actual function is unknown. At the end of mitosis, when the cell enters G1-phase, HI and H3 are rapidly dephosphorylated. The long term objective of this application is to understand the regulation of this phosphorylation and its function(s) with respect to chromosome condensation. nuclear envelope breakdown, and other processes of mitosis. The project could also shed light on protein phosphorylations in general as mechanisms of cellular and/or metabolic control. These long term goals have obvious implications for the understanding of cancer and other disease states in which normal cell cycle and/or metabolic control are disrupted. The specific aim of this project is to characterize and begin to purify the phosphohistone phosphatase which dephosphorylates H1 and H3 (and possibly other proteins) at the end of mitosis in vivo and in isolated metaphase chromosomes in vitro. This enzyme has not heretofore been investigated. An assay will be developed using the enzyme's natural substrate: the phosphoproteins in intact chromosomes. Isolated HeLa metaphase chromosome clusters will be labelled with P-32 in vitro using delta-labelled ATP and the endogenous kinase. The endogenous phosphatase will be inactivated with sulfhydryl reagents. The phosphatase (from other HeLa cells) will be assayed by measuring P-32 released from HI and H3. Using this assay, the phosphatase will be characterized with respect to optimal conditions for activity, location within the cell, conditions for extraction and activity throughout the cell cycle. Extracts of HeLa cells will be fractionate by various standard biochemical techniques in order to further characterize the enzyme and determine the feasibility of purifying it.